This application represents a request for an extension of this MERIT Award. The central hypothesis of this award is that glutathione peroxidase-3 (GPx-3) is a principal determinant of extracellular hydrogen peroxide flux from the endothelial cell, and a key regulator of the transmemrane cycling and intracellular actions of hydrogen peroxide. The key scientific accomplishments of this first phase of the award include the identification of essential regulatory determinants of GPx-3, a selenoprotein with complex and unique translational requirements; demonstration of the cofactor activation of GPx-3 catalysis by heparin via a unique polyanionic glycosaminoglycan binding site in the solvent-exposed carboxyterminal tail; the development of a GPx-3 null mouse and the characterization of its vascular and endothelial dysfunction, including a propensity to platelet-dependent thrombosis, the latter recapitulating our work in human populations of subjects with arterial ischemic or cerebral venous strokes; the identification of plasma membrane channels and transporters for hydrogen peroxide and nitric oxide; and the demonstration that mitochondria are a key source of hydrogen peroxide that can be modulated by GPx-3's intracellular counterpart, GPx-1, as well as a unique microRNA, miR210, in part responsible for the Pasteur effect. In this extension of the award, we plan to continue to pursue the same central hypothesis, but will do so in the setting of hypoxia because of the differential effect of hypoxia on GPx-3 (increases) and GPx-1 (decreases), and because of the greater role played by uncoupled mitochondria on hydrogen peroxide production under hypoxic compared with normoxic conditions. We will pursue this goal via three specific aims. First, we will examine the effect of hypoxia on GPx-3 expression, selenium uptake, and the selenoprotein synthesis hierarchy (in renal tubular cells, the primary source of plasma GPx-3 in mammals). Second, we will examine the interactions between GPx-3 and GPx-1 on endothelial hydrogen peroxide production under hypoxic conditions and its consequences for hydrogen peroxide-dependent endothelial signaling. Third, we wll examine the effects of GPx-3 on endothelial function in vivo under hypoxic conditions. These studies should complement prior work conducted during this award, and provide additional useful insights into the role of GPx-3 in the adaptive response to hypoxic stress In the endothelium.